Highly Sensitive Low-Background Fluorescent Probes for Imaging of Nitric Oxide in Cells and Tissues

Abstract

Small-molecule fluorescent probes in combination with fluorescent microscopy can be a powerful tool to provide real-time detection and high spatiotemporal resolution of transient molecules in cells and bodies. For the design of fluorescent probes for transient molecule imaging, high detection sensitivity is crucial. In this report, two new fluorescent probes, 8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-H) and 8-(3,4-diaminophenyl)-1,7-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-M), have been developed for nitric oxide (NO) imaging. The detection sensitivity has been efficiently improved by use of these probes through increasing NO detection signals and decreasing background fluorescence. Fluorescence in the far-red region is enhanced by 400- and 550-fold after reaction with NO is achieved and remains stable for at least 24 h under the irradiation of xenon lamp. Excitation and emission wavelengths longer than 600 nm and excellent intracellular retention of these probes and their NO products create dark background inside and outside cells and tissues. What is more, the excellent intracellular retention of these compounds is obtained by their strong lipophilicity, which is a novel design concept diametrically opposite to the traditional approaches. The high sensitivity and dark background make DANPBO-H and DANPBO-M competitive for NO imaging in cells and tissues. The lipophilicity-based intracellular retention mechanism as a design strategy has great potential in the development of fluorescent probes for bioimaging.

CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum

“…39,40 We note that this transportable design element is reminiscent of the 1,2-diaminobenzene-based trigger for nitric oxide sensing that has been integrated into myriad dye scaffolds. 41–44 In addition, the incorporation of zwitterionic sulfobetaine tails, which significantly outperformed both oligoethylene glycol or sulfated modifications in our hands, may be a general strategy for increasing the performance of PeT-based probes in complex environments. 45 These modifications prevent probe access to the interior of living cells, but the cytotoxicity of copper catalysts likewise limits reactions with linear alkyne-functionalized biomolecules to fixed samples or the live cell surface.…”

Near-infrared emission of dibenzoxanthenium and its application in the design of nitric oxide probes

“…The vast majority of the reported nitric oxide probes were prepared from the corresponding nitrobenzyl compounds, with the final products being obtained by reduction; 11,15 however, the method was not effective in this case since the dibenzoxanthenium will be reduced to colorless solutions. The synthetic route used for the construction of the dibenzoxanthenium compounds evaluated in this study is shown in Scheme 1.…”

“…8 The development of a new strategy for the preparation of benzoxanthenium compounds bearing diaminophenyl groups would therefore provide access to a novel series of functional dyes with diaminophenyl-benzoxanthenium photoinduced electron transfer (PET) abilities. It was envisaged that a 3,4-diaminophenyl group could be used as a suitable moiety for the development of benzoxanthenium compounds bearing diaminophenyl groups, because this group has been used previously as a reactive unit and applied to the detection of nitric oxide in probes based on some fluorophores; [9][10][11] and only one NIR nitric oxide probe based on polymethylene cyanine worked at pH 6-11 under neutral alkaline conditions. 12 Given that anilines can be protonated under acidic conditions, there is a strong need for the development of pH-independent 13 NIR probes 14 for the detection of nitric oxide that could be used to complement existing probes.…”

Development of a novel lysosome-targetable time-gated luminescence probe for ratiometric and luminescence lifetime detection of nitric oxide in vivo

“…7–9 A variety of fluorescence probes for NO have been developed, which have remarkably enriched our knowledge on NO homeostasis and its significant roles in biological processes. 10–13 Unfortunately, few of them were suitable for the quantitative detection of NO in subcellular lysosomes hitherto. One of the challenges is the lack of organelle-specificity, which results in the signal disturbance from cytosol and other organelles.…”